Infectious diseases are a leading cause of death worldwide and account for more than 13 million deaths annually including nearly two-thirds of all childhood mortality at less than 5 years of age. There is serious concern regarding new and re-emerging infectious diseases, in which effective therapies are lacking (World Health Organization reports 1999, 2012 and 2014). Antimicrobial resistance is escalating and affects a very broad range of human diseases including tuberculosis, cholera, malaria, and AIDS. Of particular concern is the number of human pathogens developing multidrug resistance to conventional antibiotics and it is estimated that the burden of resistance will surpass that of e.g. cervical cancer (de Kraker M E A. et al, PLoS Med. 2011; e1001104). The introduction of new, more potent derivatives of existing antibiotics provides only temporary solutions, since existing resistance mechanisms rapidly adapt to accommodate the new derivatives (Theuretzbacher U. Curr. Opin. Pharmacol. 2011: 11:433-438). Although resistant Gram-positive bacteria pose a significant threat, the emergence of multidrug resistant (MDR) strains of common Gram-negative pathogens such as Escherichia coli are of special concern. Pan-resistance or extreme drug resistance are now commonly used terms to describe clinically important isolates of Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae that are resistant to virtually all antibiotics (Patel et al, Front. Microbiol. 2013:4:48). Unfortunately there are few, if any, antimicrobial agents effective against Gram-negative bacteria either in or entering phase 1 clinical trials that will address this critical need (Butler M S. et al, J. Antibiotics 2013:66:571-591).
Probably the most important antibiotic resistance mechanisms in terms of distribution and clinical relevance are β-lactamases (Bush K. et al, Annu. Rev. Microbiol. 2011: 65:455-478). β-lactamases are enzymes that hydrolyse β-lactam antibiotics compromising the efficacies of β-lactams our largest group and mainstay of antimicrobial chemotherapy for >70 years. Clearly, there is a need for inhibitors directed against these classes of enzymes that will restore the activity of their substrates—antibiotics that are cheap, non-toxic and normally effective. Serine β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) have been a phenomenal success in extending the therapeutic life of β-lactam antibiotics and are also employed as diagnostic tools in clinical microbiological laboratories worldwide. In contrast, there is no clinical inhibitor available for metallo-β-lactamases (MBLs; Drawz et al., Antimicrob. Agents Chemother. 2014:58:1835-1846). The latter has now become one of the most clinically important families of β-lactamases showing global dissemination.
MBLs belong to a large group of proteins only found in bacteria, and like penicillin-binding proteins (PBPs) have the ability to interact with β-lactams. Examples of PBPs and enzymes that bind β-lactams are MBLs, serine β-lactamase-like protein (LACTB), D,D-transferase, D-Ala(D,D)-carboxypeptidase, the D-Alanyl-D-alanine Dipeptidases VanA, VanX, VanY and others, as reviewed by Sauvage E. et al in FEMS Microbiol Rev 32 (2008): 234-258. This class of proteins is only found in bacterial biology. Examples of compounds having affinity for PBPs are β-lactam antibiotics. β-Lactams have been the historical anchor of antibacterial chemotherapy and include penicillins, cephalosporins, monobactams and carbapenems (Bush K. et al, Annu. Rev. Microbiol. 2011: 65:455-478). The mechanism of action for the β-lactam antibiotics is that they mimic the small dipeptide D-Ala-D-Ala that the bacteria use to crosslink peptidoglycans in the bacterial wall, covalently destroying enzymes that use D-Ala-D-Ala as their substrate. β-lactamases are the most prevalent and clinically important resistance mechanism inactivating β-lactams by hydrolysis. They are classified according to sequence criteria (Ambler class A, B, C and D) and can be structurally grouped into two super families; the serine β-lactamases (class A, C, and D) and MBLs (class B). In contrast to the serine β-lactamases, which are characterized by a serine moiety in the active site, MBLs require divalent cations, usually zinc, as a metal co-factor of enzyme activity (Palzkill T. Ann. N.Y. Acad. Sci. 2013:91-404). MBLs are emerging as one of the most clinically important family of β-lactamases (Patel et al, Front. Microbiol. 2013:4:48; Walsh et al, Int. J. Antimicrob. Agents 2010:S8-S14) for the following reasons:
(i) Acquired MBL genes are associated with mobile genetic elements that often carry other resistance genes as gene clusters (associated with IS and ISCR elements) and/or integron arrays resulting in multi-drug resistant isolates. In many parts of the world such elements are now commonly observed in clinical important pathogens such as P. aeruginosa, A. baumannii, and several Enterobacteriaceae geni. Thus, the plasticity and dissemination of these highly mobile resistance gene clusters severely compromises existing therapeutic regimes.
(ii) The hydrolytic spectrum of MBLs is one of the broadest of all β-lactamases and includes nearly all β-lactams except the monobactams (aztreonam). Accordingly, clinical isolates possessing MBLs are invariably resistant to all β-lactam antibiotics except for the monobactams. However, virtually all MBL positive bacteria carry additional β-lactamases that can hydrolyze aztreonam and therefore this therapy is not recommended.
The clinically most important MBLs, the IMP-, VIM- and NDM-groups, are now widespread in a variety of Gram-negative species. In particular, VIM- and NDM-enzymes have emerged as the dominant MBLs. The unprecedented global dissemination of NDM highlights the enormity of the problem. Since the first report in 2008, NDM has been identified in Australia, Africa, North-America, Asia and many European countries (Johnson A P. et al, J. Med. Microbiol. 2013:62:499-513). Worryingly, NDM is found in numerous Gram-negative species and in the environment (Walsh T R. et al, Lancet Infect. Dis. 2011: 11:355-362).
Successful inhibitors of class A serine β-lactamases are clinically available, but lack inhibitory activity against MBLs (Drawz S M. et al, Clin. Microbiol. Rev. 2010:23:160-201). Inspired by the commercial success of the paradigm Augmentin (clavulanic acid—a suicide substrate for serine β-lactamases—and amoxicillin) several research groups have focused on similar approaches to develop inhibitors, but yet no molecules that combine potency with activity against multiple MBL targets have reached clinical trials (Drawz S M. et al. Antimicrob. Agents Chemother. 2014). For the three clinically most threatening MBLs—the IMP, NDM and VIM groups—most inhibitors are reported for IMP-1, while few inhibitors are found for VIM-2 and NDM. For NDM, a fungal natural product, aspergillomarasmine A has been identified as an MBL inhibitor and shown in vivo activity in mouse models (King A M. et al. Nature 2014:510:503-506). However, relatively high doses of aspergillomarasmine A are required to reverse carbapenem resistance. Other therapeutic options (Martinez, Future Med. Chem. 2012) include the use of tri-β-lactam therapy incorporating a monobactam; however, the MICs are not impressive and their in vivo activity is severely compromised by the bacterial inoculum (Page et al., Antimicrob. Agents Chemother 2011).
Thus, there is a strong clinical need for an MBL inhibitor. The majority of potent inhibitors of IMP-1 contain a thiol-carboxylate or a dicarboxylate pharmacophore, while among VIM-2 inhibitors the thiocarboxylates are the most dominant. The only inhibitors, which target IMP-1 and VIM-2 simultaneously, are found among these structural motifs. However, the recently characterized maleic acid derivatives lack broad spectrum inhibition i.e. show poor inhibition against the clinically important VIM and NDM-group enzymes. Nevertheless, some structurally very different inhibitors selectively targeting VIM-2 in favour of IMP-1 have been reported (Weide et al, Med. Chem. Lett. 2010). Few studies have evaluated the inhibitor efficiency in extensive whole cell assays and, even less so, in vivo efficacy in animal models. The majority of inhibitors heavily rely on monodentate zinc-binding groups like a thiol or a carboxylate group complexing the enzymatically bound zinc ions. Interestingly, typical bidentate zinc-binding groups successfully applied in the design of inhibitors of other zinc-containing metalloproteins like hydroxamates are—with one exception—not reported. Comparison of the available structures reveals that all inhibitors substitute the bridging hydroxide in dinuclear zinc MBLs by a heteroatom e.g. S or O.
Clinically important bacteria are well known in the prior art—see e.g. Wikipedia. Especially, P. aeruginosa causes serious problem because of its conspicuous multidrug resistance. So called scavengers, e.g. sulbactam, clavulanic acid, and tazobactam which are used as β-lactamase inhibitors in general are effective against serine-β-lactamase having serine as an active centre, but these drugs exhibit less or no inhibiting effect on MBLs. Therefore, the need for new MBL inhibitors becomes increasingly important. Important β-lactam antibiotics are the penicillin, cephlosporin and carbapenem classes. Many compounds have been reported as having MBL inhibiting activities. In WO 98/117639, WO 97/30027, WO 98/40056, WO 98/39311, and WO 97/110225, a class of β-thiopropionyl-amino acid derivatives has been described as the inhibitors against the MBLs. Other compound classes that may act as MBL inhibitors are thioesters (Biol. Pharm. Bull. 1997, 20, 1136; FEMS Microbiology Letters 1997, 157, 171; Antimicrob. Agents Chemother. 1997, 41, 135; Chem. Commun. 1998, 1609; Biochem. 1.1998, 331, 703; WO 00/076962) and succinic acid derivatives (WO 01/030148 and WO 01/030149).
The use of metal chelators against virus or bacteria has been well described in the prior art, e.g. in WO 2011/63394, WO 2004/71425, WO 2006/109069, WO 2001/60349, U.S. Pat. No. 6,410,570, US 2003/0225155, WO 2006/43153 and WO 2006/43153. The person skilled in the art will appreciate the variety of substances described in prior art, capable of chelating metal ions, and will select an appropriate chelator for different purposes. Examples are chelators comprising variations of amino groups and hydroxyl groups, e.g. 1,10-phenanthroline, clioquinol, 1,2-dimethyl-3-hydroxy-4-pyridinone (DMHP), 1,2-diethyl-3-hydroxy-4-pyridinone (DEHP), deferasirox, chelators comprising variations of amino groups, hydroxyl groups and carboxylic acid groups, e.g. ethylenediaminetetra-acetic acid (EDTA), ethylenediamine-N,N′-diacetic-N,N′-di-B-propionic acid (EDPA), diethylenetriamine pentaacetic acid (DTPA), trans-1,2-cyclohexane-diamine-N,N,N′,N′-tetraacetic acid (CyDTA), carnosine, dihydroxyethylglycine (DHEG),1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic (DTPA-OH), ethylenediamine-N,N′-diacetic acid (EDDA), ethylenediamine-N,N′-dipropionic acid (EDDP), N-hydroxy-ethylenediamine-N,N′,N′-triacetic acid (EDTA-OH), N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED), hexamethylene-1,6-diaminetetraacetic acid (HDTA), hydroxyethyliminodiacetic acid (HIDA), iminodiacetic acid (IDA), Methyl-EDTA, nitrilotriacetic acid (NTA), nitrilotripropionic acid (NTP), triethylenetetraaminehexaacetic acid (TTHA), ethylenediamine-di(O-hydroxyphenylacetic acid) (EDDHA), ethyleneglycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), trans-1,2-cyclohexanediaminetetraacetic acid (CDTA), N-(2-hydroxyethyl) ethylenedinitrilotriacetic acid (HEDTA), N-(2-hydroxyethyl) iminodiacetic acid (HEIDA), citric acid, 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11] pentatriacontane (O-Bistren), penicillamine, chelators also comprising sulfur or phosphorus, e.g. diethyldithiocarbamate(DEDTC), 2,3-dimercapto-1-propanesulfonic acid (DMPS), ethylmaltol (EM), 4-(6-Methoxy-8-quinaldinyl-aminosulfonyl)benzoic acid potassium salt (TFLZn), dithiozone, N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), ethylenediamine-N,N′-bis(methylphosphonic) acid (EDDPO), ethylenediaminetetra(methylenephosphonic) acid (EDTPO), nitrilotrimethylenphosphonic acid (NTPO), dimercaptosuccinic acid (DMSA), deferoxamine, dimercaprol, dimercaptosuccinic acid, and etidronic acid. However, these chelators are known in the prior art, e.g. in Smith, R. M.; Martell, A. E. NIST Critically Selected Stability Constants of Metal Complexes, Version 2.0; U.S. Department of Commerce: Gaithersburg, Md., 1995, as non-selective chelating agents with relatively low ability to discriminate between different metal ions. Thus, they may simultaneously have the ability to bind essential metal ions like zinc, iron, copper, nickel, cobalt, manganese and other metal ions essential in most enzymes in nature, e.g. in bacteria and mammals. This lack of selectivity may lead to undesired toxicity and other biological effects when treating specific infections by a target organism in a host organism, e.g. when it is desirable to affect only specific microorganisms whilst the toxicological effect on the host organism or other species which are not a target for the treatment is low.
A key element necessary for normal life of a bacterial cell is the homeostasis of zinc. Zinc is the second most abundant transition metal in the human body and is responsible for the catalytic function and structural stability of over 6000 enzymes and proteins (Bertini, et al, Journal of Inorganic Biochemistry 111 (2012) 150-156). Manipulation of the freely available zinc in a cell has been shown to affect a great range of diseases and conditions (Que et al, J. Chem. Rev. (Washington, D.C., U. S.) 2008, 108, 1517-1549; Peterson, et al Mol. Biol. 2009, 388, 144-158; BarKalifa et al, Eur. J. Pharmacol. 2009, 618, 15-2; Maret et al, Mol. Med. 2007, 13, 371-375). The concentration of free zinc varies in biological tissues depending on zinc buffering capacity. In PC12, HeLa, and HT-29 cell lines, as well as in primary cultures of cardiac myocytes and neurons in vitro, the concentration of free zinc has been determined to be approximately 5 nM (Bozym et al, Exp. Biol. Med. 2010, 235, 741-750). Zinc is a soft metal, mainly found in the nature as salpherite or zinc sulfite, with Zn oxidation state+2 (Emsley, J. “Zinc”. Nature's Building Blocks: An A-Z Guide to the Elements; Oxford University Press: New York, 2001). In order to chelate zinc selectively and with high efficacy, the use of soft basic ligands are needed. The lipophilic zinc chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), has been reported to display many interesting biological activities due to its zinc chelating properties as well as its lipophilic character, allowing cell-membrane penetrating abilities (Donadelli et al, J. Cell. Biochem. 2008: 104, 202-212). In the TPEN molecule, the zinc-binding ability is based on the binding of two or more units of the picoylamine unit (see Scheme 1).

Other technologies employ sulfur based ligands, also yielding high selectivity for zinc compared to other endogenous metal ions (Zhang et al, Tetrahedron 2013, 69, 15-21). Zinc sensing in bacteria is carried out by regulators of different families, including SmtB/ArsR, MerR, TetR, MarR, and the Fur family (Napolitano et al, Journal of Bacteriology 2012, p. 2426-2436). X-ray structures of MBLs indicate a tightly bound zinc ion at the active site, and often the structures reveal two Zn atoms at the catalytic site, one tight and one more loosely bound (Palzkill T. Ann. N.Y. Acad. Sci. 2013:91-404). Subclass B1 is the largest MBL family, including the majority of enzymes such as the IMPs, VIMs and NDMs. The B1 enzymes feature a conserved Zn(II) binding motif and there are two Zn2+ at the active site of B1 enzymes, with Zn1 bound by three His residues and Zn2 by one His, one Cys and one Asp. In addition, there is a bridging water or hydroxide ligand between the two Zn ions, which is believed to be the nucleophile that hydrolyses the substrate, e.g. the β-lactam antibiotic. The zinc ions are essential for the activity of the enzymes. In some embodiments the MBL inhibitors contain a Zn(II) binding group that can interact with the central metal ion(s) strongly. In the prior art, zinc chelators have been described as inhibitors of multiple diseases simultaneously, e.g. in WO 2006/117660, WO 2001/60349 or U.S. Pat. No. 6,410,570). Zinc chelators have also been suggested as antibacterial agents, e.g. in WO 2009/140215, as inhibitors of biofilm formation, e.g. in WO 2011/63394 and WO 2009/155088, or as antiviral agents, e.g. in WO 2004/71425 and WO 2006/43153. Attractive bacterial zinc-dependent targets involved in resistance mechanisms are known in the prior art to be inhibited by zinc chelating agents. Three examples are the tightly regulated bacterial zinc uptake system Zur (Ellison et al in PLOS ONE 8 (2013), e75389), biofilms (Conrady et al, PNAS (2008) 105 (49), 19456-19461) and the peptidase HmrA in MRSA. All these targets are inhibited by state of the art zinc chelators like TPEN, EDTA or the phenanthrolines. However, these agents do not possess specific affinity for bacterial biology, and are also toxic to mammals. Another example of vital zinc-dependent machineries is bacterial use of the efflux pumps (Nikaido, et al, FEMS Microbiology Reviews 36 (2012) 340-363), adapting almost all modern antibiotics as substrates, excreting them from the bacterial cell. Yet another example is the zinc-dependent deacetylase LpxC (Liang et al, J. Med. Chem. 2013, 56, 6954-6966). Another example is the highly zinc-dependent bacterial ribosomal function (Graham et el, J. Biol. Chem. 284 (2009) pp. 18377-18389). The latter uses 8 zinc atoms on the 70S ribosomal unit.
However, in these descriptions, there is no guidance on how to obtain selective effects of the zinc chelation therapy in the target species whilst a host organism is affected at a low enough level to avoid issues related to, for example, toxicity. Thus there is a medical need for zinc chelating agents with a low toxicity in a host organism, whilst also having a selective effect on the target organism.